黏液型和非黏液型铜绿假单胞菌生物被膜形成及藻酸盐基因的表达差异

The investigation of biofilm formation,alginate biosynthetic genes expression and sequence of Pseudomonas aeruginosa

目的研究黏液型铜绿假单胞菌PA17和非黏液型铜绿假单胞菌PA01的生物被膜形成及藻酸盐合成基因algD的表达,并对藻酸盐合成调节基因mucA进行序列分析。方法采用改良平板培养法建立PA17和PA01的生物被膜模型；半定量逆转录-聚合酶链反应(RT-PCR)测定浮游状态及生物被膜形成不同时间点藻酸盐合成基因algD的表达情况,并进行软件分析；PCR方法扩增藻酸盐合成调节基因mucA并测序。结果PA17和PA01分别于第6天和第3天形成成熟生物被膜,均为薄膜状。浮游状态时,PA17的algD表达较PA01高7倍。生物被膜形成前期,PA17和PA01的algD表达水平相近；algD在成熟生物被膜形成时表达水平最高,PA17的algD表达稍高于PA01。PA17的mucA基因第166～333位核苷酸缺失,第342位A→G；PA01的mucA基因序列与基因库中公布的序列完全相同。结论PA17含新型mucA基因突变,可能是造成生物被膜形成时PA17与PA01的藻酸盐合成基因algD的表达差异较浮游状态时低,进而造成两者生物被膜形态相似的原因。

Objective To investigate the biofilm formation, the alginate biosynthetic gene expression and analyze the mucA gene sequence of mucoid Pseudomonas aeruginosa PA17 and nonmucoid Pseudomonas aeruginosa PA01. Methods The modified plate culture method was used to establish the biofilm model in vitro. Semi quantitative RT PCR was used to determine the expression level of algD in planktonic condition and during the formation of biofilm. The mucA gene of PAl7 and PA01 was amplified and the products were sequenced. Results PA17 biofilm was mature at 6th day, and PA01 biofilm was mature at 3rd day. The structures of the biofilms were both like pellicle. In planktonic condition, the algD expression of PA17 was higher than PA01 ; in biofilm formation, the algD expression was maximal when the biofilm was mature. There was a 166～333 deletion mutation and 342A→G in mucA gene of PAl7, the mucA gene of PA01 was the same with the sequence of Genbank. Conclusions The mucA gene mutation of PA17 was a new type, which maybe the reason for the little expression difference of algD between PA17 and PA01 during the biofilm formation than it in planctonic condition and the same structure of PA17 and PA01 biofilm.